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Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung's disease and total colonic aganglionosis
Neuroscience Letters 451 (2009) 208–211
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung’s disease and total colonic aganglionosis Hefeng Wang a , Yuhua Zhang b , Wei Liu a , Rongde Wu a,∗ , Xinguo Chen a , Linuan Gu a , Bin Wei b , Yingmao Gao b a b
Department of Pediatric Surgery, Shandong Provincial Hospital of Shandong University, 324 Jingwu Weiqi Road, Jinan, Shandong Province, China Department of Histology and Embryology, School of Medicine, Shandong University, Jinan, Shandong Province, China
a r t i c l e
i n f o
Article history: Received 19 October 2008 Received in revised form 6 January 2009 Accepted 7 January 2009 Keywords: c-Kit Enteric plexus Hirschsprung’s disease Interstitial cells of Cajal Total colonic aganglionosis
a b s t r a c t Interstitial cells of Cajal (ICCs) play a key role in regulating gastrointestinal tract motility. The pathophysiological basis of colonic aperistalsis in Hirschsprung’s disease (HD) is still not fully understood. Many studies reported that decreased numbers or disrupted networks of ICCs were associated with HD. Little information is available on the distribution of different subtypes of ICCs in HD. The aim of this study was to determine the alterations in density of different subtypes of ICC in colonic specimens of patients with total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. ICCs were visualized in frozen sections by c-Kit (CD117) fluorescent staining. In the control colon, c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), along the submucosal surface of the circular muscle layer (IC-SM) and in the circular and longitudinal muscle layer (IC-IM). In the aganglionic region of the colon of the patients affected by HD, the number of ICCs (especially IC-IM and IC-SM) was markedly reduced and IC-MY networks were disrupted. Nearly total lack of three subtypes of ICCs was observed in the TCA specimens. This study demonstrated the altered distribution of different subtypes of ICCs in the resected colon of patients with recto-sigmoid HD and TCA. These findings suggest that the reduction of each subtype of ICCs may play an important role in the etiology of HD. © 2009 Elsevier Ireland Ltd. All rights reserved.
The normal motility in the gastrointestinal tract is known to depend on the enteric nervous system, the smooth muscle and the interstitial cells of Cajal (ICCs). The ICCs are pacemaker cells, which generate slow waves and facilitate active propagation of electrical events and mediate neurotransmission of the gastrointestinal tract [18]. Three subtypes of ICCs were identified based on anatomical location within the human colon: ICCs in the myenteric plexus region (IC-MY) and along the submucosal surface of the circular muscle layer (IC-SM), and in the circular and longitudinal muscle layer (IC-IM). Each subtype of ICCs has special function [9,16,24]. Hirschsprung’s disease (HD) is a functional intestinal obstruction with an incidence of 1/5000 live births. Recto-sigmoid HD occurs in over 75% of all patients with HD, when the aganglionic segment does not extend beyond the upper sigmoid, and total colonic aganglionosis (TCA) is reported to occur in 2–14% of patients with HD, when aganglionosis extends within 30 cm proximal to the terminal ileum [3,10,18,17]. Up to today, the absolute histomorphological criterion for diagnosis of HD is that there is a lack of ganglia. Many
∗ Corresponding author. Tel.: +86 531 85186338; fax: +86 531 87061968. E-mail address: [email protected] (R. Wu). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.01.015
studies using immunohistochemistry and whole-mount preparation showed that decreased numbers or disrupted networks of ICCs were associated with HD [21,25]. However, there is little information available on the distribution of different subtypes of ICCs in HD. In our experiment, we used immunohistochemistry to compare the changes of different subtypes of ICCs in the recto-sigmoid HD and TCA. Aganglionic colon specimens were obtained from five children with TCA (age range from 2 months to 23 months, mean age 7.8 months, 4 boys and 1 girl) and sixteen with recto-sigmoid HD (age range from 1 month to 73 months, mean age 26.9 months, 14 boys and 2 girls) taken during operation. Normal colon specimens were collected as control specimens from seven children (age range from 5 months to 26 months, mean age 11.6 months, 5 boys and 2 girls) at the time of bladder augmentation. All colon specimens obtained immediately after resection were cut into blocks (10 mm length) and snap frozen in liquid nitrogen until use. Frozen serial sections (10 m thick) were cut on a cryostat and mounted onto slides coated with 0.1% poly-l-lysine. Sections were then fixed in ice-cold absolute acetone for 10 min and stored at 4 ◦ C. Our procedures were in accordance with ethical standards as formulated in the Helsinki Declaration of 1975 (revised 1983).
H. Wang et al. / Neuroscience Letters 451 (2009) 208–211
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Fig. 1. Distribution of ICCs in the control colon. (A) The ICCs were present in the longitudinal muscle layer, the circular muscle layer and around the myenteric plexus. (B) The processes of ICCs located in the CM are parallel to the smooth muscle cells. (C) c-Kit positive ICCs around the myenteric plexus between longitudinal and circular muscle layers. They formed a dense network. (D) c-Kit positive ICCs along the submucosal surface of the circular muscle layer (arrowheads) and in the circular muscle layers (arrows). (E and F) At higher magnification, details of two types of ICCs: multipolar and bipolar, located around the myenteric plexus and in the circular muscle layers. SM, submucosa; CM, circular muscle; LM, longitudinal muscle; Scale bar is 500 m in panels A and B; 200 m in panels C and D and 100 m in panels E and F.
The sections were washed for 10 min in phosphate buffered saline (PBS; 0.05 mol/L, pH 7.4, with 0.3% Triton X-100), blocked in 10% goat serum for 30 min at room temperature and incubated with monoclonal mouse anti-human c-Kit (1:100, Biolegend, San Diego) at 4 ◦ C overnight. Sections then were incubated at room temperature for 1 h with TRITC-conjugated goat anti-mouse IgG (Molecular Probes, The Netherlands) diluted 1:100 in PBS and cover-slipped with a fluorescence mounting medium (DAKO, Danmark). Negative controls were checked by omitting the primary antibody. Images were acquired on a fluorescence microscope (Olympus, Tokyo, Japan). Morphometric analysis was performed to determine the distribution and density of c-Kit-positive ICCs in patients and controls. The different subtypes of ICCs were divided into three anatomical regions: IC-SM, IC-IM and IC-MY. In each of these regions, 20 fields were scanned with a ×20 objective. The area occupied by ICC was calculated with Image Processing Plus System (Media Cybernetics USA). The area of each microscopic field was 0.48 mm2 . The data obtained were expressed as mean ± SEM. One-way ANOVA was used to compare the statistical difference among the
control group, recto-sigmoid HD and TCA patients, with P < 0.05 being considered an indicator of significance. ICCs were located throughout the circular and longitudinal muscle layers, myenteric plexus regions and along the submucosal surface of the circular muscle layer (Fig. 1A and D). IC-MY formed a dense network encasing the myenteric plexus (Fig. 1C and E). IC-IM appeared as long, thin, bipolar cells with one or two processes connecting with each other (Fig. 1B and D). There were two phenotypes of c-Kit-positive ICCs: bipolar and multipolar cells (Fig. 1E and F). The percentage of the area occupied by IC-SM, IC-IM and IC-MY was 0.97%, 1.28% and 1.63%, respectively. In the aganglionic region of the colon of the patients affected by HD, the c-Kit positive aera of ICCs located in the three regions (IC-SM, IC-IM, IC-MY) was markedly decreased (Fig. 2A). Compared with processes from ICCs visualized in controls, the projections of remaining IC-MY became blunt and short (Fig. 2B). In the aganglionic region of the colon of the patients affected by TCA, there was nearly a total lack of IC-IM, IC-MY and IC-SM in all the frozen sections (Fig. 2C). In contrast to controls, the density of ICCs in these three regions (IC-SM, IC-IM, IC-MY) of aganglionic colon from TCA and recto-sigmoid patients was considerably decreased (P < 0.01,
Fig. 2. The ICCs in the aganglionic colon of patients with recto-sigmoid HD and TCA. The density of ICCs in the aganglionic colon of recto-sigmoid HD patients decreased significantly and only a few ICCs were observed around the myenteric plexus (A). At higher magnification, details of processes from the remaining ICCs (arrow) appeared to be blunt and short compared with processes from ICCs in controls (B). Absence of ICCs in submucosal layer, myenteric plexus and muscle layer of TCA sample (C). CM, circular muscle; LM, longitudinal muscle; Scale bar is 500 m in panels A and C, 100 m in panel B.
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H. Wang et al. / Neuroscience Letters 451 (2009) 208–211
Fig. 3. The percentage of area occupied by IC-MY (A), IC-IM (B), IC-SM (C) in the colon of control, recto-sigmoid HD and TCA patients.
Fig. 3). Furthermore, TCA patients had a significantly lower IC-MY density compared with recto-sigmoid HD patients (P < 0.01). There were no significant differences in IC-SM and IC-IM density between recto-sigmoid HD and TCA (P > 0.05). In the human colon, ICCs are located at the level of myenteric plexus, submucosal layer and within the longitudinal muscle layer and the circular muscle itself. Previous studies suggested that ICCs have three major functions in the gastrointestinal tract: (1) they are pacemaker cells in smooth muscle, (2) they facilitate the propagation of electrical events and (3) they mediate neurotransmission [15,22,23]. There are three subtypes of ICCs in the colon and each of them has diverse function in the colonic motility. Morphological and functional studies have suggested that IC-MY and IC-SM act as pacemakers and conductors of electrical slow waves in gastrointestinal muscles. W/W v mutant mice have deficient ICCs in the small intestinal myenteric plexus, which leads to the absence of electrical slow waves and abnormal, slow and uncoordinated motility [4]. It was reported that loss of ICCs involved in neurotransmission could be as significant in the pathophysiology of motility disorders as damage to pacemaker ICCs [1]. IC-IM is frequently located between nerve endings and smooth muscle cells, establishing synapse-like contacts and gap junctions [5]. Therefore, the role of IC-IM in enteric motor neurotransmission is particularly important. Several studies have suggested that IC-IM might be lost or damaged in a variety of motility disorders, such as pseudoobstruction [11,13], megacolon [6], and chronic constipation [8,12]. As demonstrated by studies of W/W v mice, which lack gastric ICIM, loss of these cells seriously impairs neural control of motility [2,23,22]. In this study we observed the distribution of c-Kit positive ICMY, IC-SM and IC-IM in normal colon, which was similar to previous reports [7,14,19,20]. To our knowledge, ICCs and mast cells express Kit and this might create confusion when performing a quantitative analysis. However, mast cells possess a round-shaped body and in the colon of our patients these cells were mainly located in the mucosal and submucosal layers. Therefore, they could be easily distinguished form ICCs and were excluded from the analysis of our study. In the present study, we investigated aganglionic colon of patients with recto-sigmoid HD and TCA and found a marked reduction of c-Kit positive ICCs. In recto-sigmoid HD patients, the decline of IC-IM and IC-SM was more prominent than that of ICMY. There was nearly a total absence of IC-MY, IC-IM and IC-SM in TCA specimens. The damage to pacemaker ICCs (especially IC-MY) in TCA patients was more significant than that of recto-sigmoid HD patients. The depletion of ICCs in the colon wall contributed to the inability of the smooth muscle to relax. This may partially explain the serious dysfunction of gastrointestinal tract motility in TCA. In summary, we demonstrated the distribution and density of different subtypes of ICCs within the colon of recto-sigmoid HD and TCA patients and that each subtype of ICCs was decreased in number or was even lacking. The lack or reduction of ICCs in TCA
and HD patients may lead to defective generation of pacemaker activity, thus contributing, together with the lack of neurons, to the motility dysfunction present in HD. Acknowledgement This work was supported by a grant from the Science Research Development Project of Shandong province (No. 2006GG2202028). References [1] G.E. Boeckxstaens, Understanding and controlling the enteric nervous system, Best Pract. Res. Clin. Gastroenterol. 16 (2002) 1013–1023. [2] A.J. Burns, A.E.J. Lomax, S. Torihashi, K.M. Sanders, S.M. Ward, Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 12008–12013. [3] A.G. Coran, R. Bjordal, S. Eek, O. Knutrud, The surgical management of total colonic aganglionosis and small intestinal aganglionosis, J. Pediatr. Surg. 4 (1969) 531–536. [4] T. Der-Silaphet, J. Malysz, S. Hagel, A. Larry Arsenault, J.D. Huizinga, Interstitial cells of Cajal direct normal propulsive contractile activity in the mouse small intestine, Gastroenterology 114 (1998) 724–736. [5] M.S. Faussone-Pellegrini, C. Cortesini, Ultrastructural features and localization of the interstitial cells of Cajal in the smooth muscle coat of human esophagus, J. Submicrosc. Cytol. 17 (1985) 187–197. [6] M.S. Faussone-Pellegrini, P. Fociani, R. Buffa, G. Basilisco, Loss of interstitial cells and a fibromuscular layer on the luminal side of the colonic circular muscle presenting as megacolon in an adult patient, Gut 45 (1999) 775–779. [7] R. Hagger, S. Gharaie, C. Finlayson, D. Kumar, Distribution of the interstitial cells of Cajal in the human anorectum, J. Auton. Nerv. Syst. 73 (1998) 75–79. [8] C.L. He, L. Burgart, L. Wang, J. Pemberton, T. Young-Fadok, J. Szurszewski, G. Farrugia, Decreased interstitial cell of Cajal volume in patients with slow-transit constipation, Gastroenterology 118 (2000) 14–21. [9] J.D. Huizinga, Physiology and pathophysiology of the interstitial cells of Cajal: from bench to bedside. II. Gastric motility: lessons from mutant mice on slow waves and innervations, Am. J. Physiol. Gastrointest. Liver Physiol. 281 (2001) 1129–1134. [10] K. Ikeda, S. Goto, Total colonic aganglionosis with or without small bowel involvement: an analysis of 127 patients, J. Pediatr. Surg. 21 (1971) 319–322. [11] K. Isozaki, S. Hirota, J. Miyagawa, M. Taniguchi, Y. Shinomura, Y. Matsuzawa, Deficiency of c-Kit+ cells in patients with a myopathic form of chronic idiopathic intestinal pseudoobstruction, Am. J. Gastroenterol. 92 (1997) 332–334. [12] S.E. Kenny, M.G. Connell, R.J. Rintala, C. Vaillant, D.H. Edgar, D.A. Lloyd, Abnormal colonic interstitial cells of Cajal in children with anorectal malformations, J. Pediatr. Surg. 33 (1998) 130–132. [13] S.E. Kenny, J.M. Vanderwinden, R.J. Rintala, M.G. Connell, D.A. Lloyd, J.J. Vanderhaegen, M.H. De Laet, Delayed maturation of the interstitial cells of Cajal: a new diagnosis for transient neonatal pseudoobstruction. Report of two cases, J. Pediatr. Surg. 33 (1998) 94–98. [14] G.L. Lyford, C.L. He, E. Soffer, T.L. Hull, S.A. Strong, A.J. Senagore, L.J. Burgart, T. Yong-Fadok, J.H. Szurszewski, G. Farrugia, Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation, Gut 51 (2002) 496–501. [15] K.M. Sanders, A case of interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract, Gastroenterology 111 (1996) 429–515. [16] K.M. Sander, T. Ordog, S.M. Ward, Physiology and pathophysiology of the interstitial cells of Cajal: from bench to bedside. IV. Genetic and animal models of GI motility disorders caused by loss of interstitial cells of Cajal, Am. J. Physiol. Gastrointest. Liver Physiol. 282 (2002) 747–756. [17] V. Solari, A.P. Piotrowska, P. Puri, Histopathological differences between rectosigmoid Hirschsprung’s disease and total colonic aganglionosis, Pediatr. Surg. Int. 19 (2003) 349–354. [18] L.R. Soltero-Harrington, G.R. Rinaldi, L.W. Able, Total colonic aganglionosis of the colon, recognition and management, J. Pediatr. Surg. 21 (1969) 330–335.
H. Wang et al. / Neuroscience Letters 451 (2009) 208–211 [19] W.D. Tong, B.H. Liu, L.Y. Zhang, S.B. Zhang, Y. Lei, Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation, Int. J. Colorectal Dis. 19 (2004) 467–473. [20] S. Torihashi, M. Horisawa, Y. Watanabe, c-Kit immunoreactive interstitial cells in the human gastrointestinal tract, J. Auton. Nerv. Syst. 75 (1999) 38–50. [21] J.M. Vanderwinden, J.J. Rumessen, H. Liu, D. Descamps, M.H. De Laet, J.J. Vanderwinden, Interstitial cells of Cajal in human colon and in Hirschsprung’s disease, Gastroenterology 111 (1996) 901–910. [22] S.M. Ward, E.A.H. Beckett, X.Y. Wang, F. Baker, M. Khoyi, K.M. Sanders, Interstitial cells of Cajal mediated cholinergic neurotransmission from enteric motor neurons, J. Neurosci. 15 (2000) 1393–1403.
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[23] S.M. Ward, G. Morris, L. Reese, X.Y. Wang, K.M. Sanders, Interstitial cells of Cajal mediated enteric inhibitory neurotransmission in the lower oesophagus and pyloric sphincters, Gastroenterology 115 (1998) 314–329. [24] S.M. Ward, K.M. Sanders, Physiology and pathophysiology of the interstitial cells of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks, Am. J. Physiol. Gastrointest. Liver Physiol. 281 (2001) 602–611. [25] A. Yamataka, Y. Kato, D. Tibboel, Y. Murata, N. Sueyoshi, T. Fujimoto, H. Nishiye, T. Miyano, A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung’s disease, J. Pediatr. Surg. 30 (3) (1995) 441–444.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung’s disease and total colonic aganglionosis Hefeng Wang a , Yuhua Zhang b , Wei Liu a , Rongde Wu a,∗ , Xinguo Chen a , Linuan Gu a , Bin Wei b , Yingmao Gao b a b
Department of Pediatric Surgery, Shandong Provincial Hospital of Shandong University, 324 Jingwu Weiqi Road, Jinan, Shandong Province, China Department of Histology and Embryology, School of Medicine, Shandong University, Jinan, Shandong Province, China
a r t i c l e
i n f o
Article history: Received 19 October 2008 Received in revised form 6 January 2009 Accepted 7 January 2009 Keywords: c-Kit Enteric plexus Hirschsprung’s disease Interstitial cells of Cajal Total colonic aganglionosis
a b s t r a c t Interstitial cells of Cajal (ICCs) play a key role in regulating gastrointestinal tract motility. The pathophysiological basis of colonic aperistalsis in Hirschsprung’s disease (HD) is still not fully understood. Many studies reported that decreased numbers or disrupted networks of ICCs were associated with HD. Little information is available on the distribution of different subtypes of ICCs in HD. The aim of this study was to determine the alterations in density of different subtypes of ICC in colonic specimens of patients with total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. ICCs were visualized in frozen sections by c-Kit (CD117) fluorescent staining. In the control colon, c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), along the submucosal surface of the circular muscle layer (IC-SM) and in the circular and longitudinal muscle layer (IC-IM). In the aganglionic region of the colon of the patients affected by HD, the number of ICCs (especially IC-IM and IC-SM) was markedly reduced and IC-MY networks were disrupted. Nearly total lack of three subtypes of ICCs was observed in the TCA specimens. This study demonstrated the altered distribution of different subtypes of ICCs in the resected colon of patients with recto-sigmoid HD and TCA. These findings suggest that the reduction of each subtype of ICCs may play an important role in the etiology of HD. © 2009 Elsevier Ireland Ltd. All rights reserved.
The normal motility in the gastrointestinal tract is known to depend on the enteric nervous system, the smooth muscle and the interstitial cells of Cajal (ICCs). The ICCs are pacemaker cells, which generate slow waves and facilitate active propagation of electrical events and mediate neurotransmission of the gastrointestinal tract [18]. Three subtypes of ICCs were identified based on anatomical location within the human colon: ICCs in the myenteric plexus region (IC-MY) and along the submucosal surface of the circular muscle layer (IC-SM), and in the circular and longitudinal muscle layer (IC-IM). Each subtype of ICCs has special function [9,16,24]. Hirschsprung’s disease (HD) is a functional intestinal obstruction with an incidence of 1/5000 live births. Recto-sigmoid HD occurs in over 75% of all patients with HD, when the aganglionic segment does not extend beyond the upper sigmoid, and total colonic aganglionosis (TCA) is reported to occur in 2–14% of patients with HD, when aganglionosis extends within 30 cm proximal to the terminal ileum [3,10,18,17]. Up to today, the absolute histomorphological criterion for diagnosis of HD is that there is a lack of ganglia. Many
∗ Corresponding author. Tel.: +86 531 85186338; fax: +86 531 87061968. E-mail address: [email protected] (R. Wu). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.01.015
studies using immunohistochemistry and whole-mount preparation showed that decreased numbers or disrupted networks of ICCs were associated with HD [21,25]. However, there is little information available on the distribution of different subtypes of ICCs in HD. In our experiment, we used immunohistochemistry to compare the changes of different subtypes of ICCs in the recto-sigmoid HD and TCA. Aganglionic colon specimens were obtained from five children with TCA (age range from 2 months to 23 months, mean age 7.8 months, 4 boys and 1 girl) and sixteen with recto-sigmoid HD (age range from 1 month to 73 months, mean age 26.9 months, 14 boys and 2 girls) taken during operation. Normal colon specimens were collected as control specimens from seven children (age range from 5 months to 26 months, mean age 11.6 months, 5 boys and 2 girls) at the time of bladder augmentation. All colon specimens obtained immediately after resection were cut into blocks (10 mm length) and snap frozen in liquid nitrogen until use. Frozen serial sections (10 m thick) were cut on a cryostat and mounted onto slides coated with 0.1% poly-l-lysine. Sections were then fixed in ice-cold absolute acetone for 10 min and stored at 4 ◦ C. Our procedures were in accordance with ethical standards as formulated in the Helsinki Declaration of 1975 (revised 1983).
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Fig. 1. Distribution of ICCs in the control colon. (A) The ICCs were present in the longitudinal muscle layer, the circular muscle layer and around the myenteric plexus. (B) The processes of ICCs located in the CM are parallel to the smooth muscle cells. (C) c-Kit positive ICCs around the myenteric plexus between longitudinal and circular muscle layers. They formed a dense network. (D) c-Kit positive ICCs along the submucosal surface of the circular muscle layer (arrowheads) and in the circular muscle layers (arrows). (E and F) At higher magnification, details of two types of ICCs: multipolar and bipolar, located around the myenteric plexus and in the circular muscle layers. SM, submucosa; CM, circular muscle; LM, longitudinal muscle; Scale bar is 500 m in panels A and B; 200 m in panels C and D and 100 m in panels E and F.
The sections were washed for 10 min in phosphate buffered saline (PBS; 0.05 mol/L, pH 7.4, with 0.3% Triton X-100), blocked in 10% goat serum for 30 min at room temperature and incubated with monoclonal mouse anti-human c-Kit (1:100, Biolegend, San Diego) at 4 ◦ C overnight. Sections then were incubated at room temperature for 1 h with TRITC-conjugated goat anti-mouse IgG (Molecular Probes, The Netherlands) diluted 1:100 in PBS and cover-slipped with a fluorescence mounting medium (DAKO, Danmark). Negative controls were checked by omitting the primary antibody. Images were acquired on a fluorescence microscope (Olympus, Tokyo, Japan). Morphometric analysis was performed to determine the distribution and density of c-Kit-positive ICCs in patients and controls. The different subtypes of ICCs were divided into three anatomical regions: IC-SM, IC-IM and IC-MY. In each of these regions, 20 fields were scanned with a ×20 objective. The area occupied by ICC was calculated with Image Processing Plus System (Media Cybernetics USA). The area of each microscopic field was 0.48 mm2 . The data obtained were expressed as mean ± SEM. One-way ANOVA was used to compare the statistical difference among the
control group, recto-sigmoid HD and TCA patients, with P < 0.05 being considered an indicator of significance. ICCs were located throughout the circular and longitudinal muscle layers, myenteric plexus regions and along the submucosal surface of the circular muscle layer (Fig. 1A and D). IC-MY formed a dense network encasing the myenteric plexus (Fig. 1C and E). IC-IM appeared as long, thin, bipolar cells with one or two processes connecting with each other (Fig. 1B and D). There were two phenotypes of c-Kit-positive ICCs: bipolar and multipolar cells (Fig. 1E and F). The percentage of the area occupied by IC-SM, IC-IM and IC-MY was 0.97%, 1.28% and 1.63%, respectively. In the aganglionic region of the colon of the patients affected by HD, the c-Kit positive aera of ICCs located in the three regions (IC-SM, IC-IM, IC-MY) was markedly decreased (Fig. 2A). Compared with processes from ICCs visualized in controls, the projections of remaining IC-MY became blunt and short (Fig. 2B). In the aganglionic region of the colon of the patients affected by TCA, there was nearly a total lack of IC-IM, IC-MY and IC-SM in all the frozen sections (Fig. 2C). In contrast to controls, the density of ICCs in these three regions (IC-SM, IC-IM, IC-MY) of aganglionic colon from TCA and recto-sigmoid patients was considerably decreased (P < 0.01,
Fig. 2. The ICCs in the aganglionic colon of patients with recto-sigmoid HD and TCA. The density of ICCs in the aganglionic colon of recto-sigmoid HD patients decreased significantly and only a few ICCs were observed around the myenteric plexus (A). At higher magnification, details of processes from the remaining ICCs (arrow) appeared to be blunt and short compared with processes from ICCs in controls (B). Absence of ICCs in submucosal layer, myenteric plexus and muscle layer of TCA sample (C). CM, circular muscle; LM, longitudinal muscle; Scale bar is 500 m in panels A and C, 100 m in panel B.
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Fig. 3. The percentage of area occupied by IC-MY (A), IC-IM (B), IC-SM (C) in the colon of control, recto-sigmoid HD and TCA patients.
Fig. 3). Furthermore, TCA patients had a significantly lower IC-MY density compared with recto-sigmoid HD patients (P < 0.01). There were no significant differences in IC-SM and IC-IM density between recto-sigmoid HD and TCA (P > 0.05). In the human colon, ICCs are located at the level of myenteric plexus, submucosal layer and within the longitudinal muscle layer and the circular muscle itself. Previous studies suggested that ICCs have three major functions in the gastrointestinal tract: (1) they are pacemaker cells in smooth muscle, (2) they facilitate the propagation of electrical events and (3) they mediate neurotransmission [15,22,23]. There are three subtypes of ICCs in the colon and each of them has diverse function in the colonic motility. Morphological and functional studies have suggested that IC-MY and IC-SM act as pacemakers and conductors of electrical slow waves in gastrointestinal muscles. W/W v mutant mice have deficient ICCs in the small intestinal myenteric plexus, which leads to the absence of electrical slow waves and abnormal, slow and uncoordinated motility [4]. It was reported that loss of ICCs involved in neurotransmission could be as significant in the pathophysiology of motility disorders as damage to pacemaker ICCs [1]. IC-IM is frequently located between nerve endings and smooth muscle cells, establishing synapse-like contacts and gap junctions [5]. Therefore, the role of IC-IM in enteric motor neurotransmission is particularly important. Several studies have suggested that IC-IM might be lost or damaged in a variety of motility disorders, such as pseudoobstruction [11,13], megacolon [6], and chronic constipation [8,12]. As demonstrated by studies of W/W v mice, which lack gastric ICIM, loss of these cells seriously impairs neural control of motility [2,23,22]. In this study we observed the distribution of c-Kit positive ICMY, IC-SM and IC-IM in normal colon, which was similar to previous reports [7,14,19,20]. To our knowledge, ICCs and mast cells express Kit and this might create confusion when performing a quantitative analysis. However, mast cells possess a round-shaped body and in the colon of our patients these cells were mainly located in the mucosal and submucosal layers. Therefore, they could be easily distinguished form ICCs and were excluded from the analysis of our study. In the present study, we investigated aganglionic colon of patients with recto-sigmoid HD and TCA and found a marked reduction of c-Kit positive ICCs. In recto-sigmoid HD patients, the decline of IC-IM and IC-SM was more prominent than that of ICMY. There was nearly a total absence of IC-MY, IC-IM and IC-SM in TCA specimens. The damage to pacemaker ICCs (especially IC-MY) in TCA patients was more significant than that of recto-sigmoid HD patients. The depletion of ICCs in the colon wall contributed to the inability of the smooth muscle to relax. This may partially explain the serious dysfunction of gastrointestinal tract motility in TCA. In summary, we demonstrated the distribution and density of different subtypes of ICCs within the colon of recto-sigmoid HD and TCA patients and that each subtype of ICCs was decreased in number or was even lacking. The lack or reduction of ICCs in TCA
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